Abstract Body: An important component of cardiac myofilament activation is the overlap structure between neighboring tropomyosin (Tm) molecules and the N-terminal extension of cardiac troponin T. Previous studies have shown that the DCM-linked tropomyosin mutation, D230N-Tm, decreases the distance across the center of the overlap resulting in a compaction in this region and a concomitant decrease in flexibility. Given the mutation-induced structural changes, we screened for molecules that could target this compaction utilizing a fully atomistic cardiac thin filament model. Z06 was identified and shown to alleviate compaction of the overlap in D230N-Tm in both our model and in fully reconstituted cardiac thin filaments. We hypothesized that directly targeting the structural changes at the overlap can improve regulatory and cardiac function in D230N-Tm mice. To first investigate if Z06 could improve myofilament activation we performed an NADH-coupled ATPase assay at varying calcium concentrations. D230N-Tm myofibrils demonstrated a reduced Hill slope (0.79±0.19) compared to its Non-transgenic (NTg) littermates (1.70±0.35) supporting a decrease in cooperativity of myofilament activation. In the presence of 10um Z06 the Hill slope of D230N-Tm increased (1.27±0.14) and was restored to NTg levels. Similarly, the addition of Z06 improved contractility parameters in isolated cardiomyocytes. D230N-Tm cells exhibited a delayed time to peak, time to peak 50% and time to baseline 50%, and the addition of 10uM Z06 was able to restore these parameters to NTg levels. Finally, we performed preclinical studies in our transgenic mice to investigate any improvement in systolic function with Z06 administration. We administered Z06 to 2-month-old D230N-Tm mice and their NTg littermates. At baseline, D230N-Tm mice exhibited a decrease in percent fractional shortening (%FS) of 18.84±0.405 compared to their NTg littermates (28.59±0.67). After four weeks of treatment we observed an improvement in %FS in D230N-Tm mice to 25.19±0.8127 with no significant changes to systolic function in NTg littermates. Future work will investigate the efficacy of Z06 as a potential late-stage treatment and assess its general applicability in other thin filament mutations, such as, D219N-Tm (HCM).